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Direct Site-Directed Photocoupling of Proteins onto Surfaces Coated with beta-Cyclodextrins

På svenska

Author

  • Rasmus L. Jensen
  • Lars W. Stade
  • Reinhard Wimmer
  • Allan Stensballe
  • Meg Duroux
  • Kim L. Larsen
  • Christer Wingren
  • Laurent Duroux

Summary, in English

A method called Dock'n'Flash was developed to offer site-specific capture and direct UVA-induced photocoupling of recombinant proteins. The method involves the tagging of recombinant proteins with photoreactive p-benzoyl-L-phenylalanine (pBpa) by genetic engineering. The photoreactive pBpa tag is used for affinity capture of the recombinant protein by beta-cyclodextrin (beta-CD), which provides hydrogen atoms to be abstracted in the photocoupling process. To exemplify the method, a recombinant, folded, and active N27pBpa mutant of cutinase from Fusarium solani pisi was produced in E. coli. Insertion of pBpa was verified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy. A inolecular dynamic simulation, with water as solvent, showed high solvent accessibility of the pBpa benzophenone group in N27pBpa-cutinase mutant. The formation of an inclusion complex between the benzophenone group of N27pBpa-cutinase and beta-CD was shown, and an apparent K-d of 1.65 mM was determined using H-1 NMR. Photocoupling of beta-CD to N27pBpa-cutinase in a 1:1 ratio, upon UVA irradiation at 360 +/- 20 nm, was shown by MALDI-TOF mass spectroscopy. UVA photoimmobilization of N27pBpa-cutinase on quartz slides coated with beta-CD was achieved from liquid or dry films by total internal reflection fluorescence (TIRF). The Dock'n'Flash method offers a solution for direct photocoupling and patterning of recombinant proteins onto surfaces with site-specific attachment,

Publishing year

2010

Language

English

Pages

11597-11604

Publication/Series

Langmuir

Volume

26

Issue

13

Document type

Journal article

Publisher

The American Chemical Society (ACS)

Topic

  • Immunology in the medical area

Status

Published

ISBN/ISSN/Other

  • ISSN: 0743-7463